Conformational Change Detection in Nonmetal Proteins by Direct Electrochemical Oxidation Using Diamond Electrodes

Abstract

In this report, we established a new electrochemical method for the detection of conformational changes in large, non-metalloproteins such as bovine serum albumin, using flow injection analysis coupled with hydrogen-terminated, boron-doped diamond electrodes. The oxidation current was used as a signal reporter in the monitoring of urea-induced BSA denaturation. In the denatured state at high urea concentrations, the electrochemical signal increased, and the amperometric responses for the oxidation potential at 1300 mV were consistent with the results of conventional methods of denaturation monitoring using fluorescence spectroscopy. The oxidation involved at least five redox-active species (cysteine, tryptophan, tyrosine, methionine, and disulfide bonds). Furthermore, the method also showed high sensitivity for quantitative analysis of protein. A linear dynamic in the concentration range 50-400 microg/mL (r(2) = 0.977) with a lower detection limit of 190 ng/mL was achieved for BSA. Direct electrochemical detection of conformation changes of proteins using BDD electrodes can be performed with advantages in terms of simplicity and sensitivity.

Modeling colloid deposition on a protein layer adsorbed to iron-oxide-coated sand

“…The molecule is flexible and amorphous, consisted of amino acids and peptide bonds (Nakanishi et al, 2001;Rogalinski et al, 2005) and has an isoelectric point of approximately pH4.7 (Chiku et al, 2008). There is a consensus that surface properties of BSA vary across the molecule giving rise to charge heterogeneity (Baier et al, 2011), along with hydrophobic and hydrophilic regions (Yoon et al, 1998).…”

“…There is a consensus that surface properties of BSA vary across the molecule giving rise to charge heterogeneity (Baier et al, 2011), along with hydrophobic and hydrophilic regions (Yoon et al, 1998). Consequently, morphological models of BSA needs simplification to certain degrees: it is described either as a globular ellipsoid of about 14 x 3.8 x 3.8 nm (Ke et al, 2009;Togashi et al, 2009), a heart-shaped solid with three different domains (Voros, 2004), or a flexible foldable polymer (Chiku et al, 2008). Among them, the ellipsoid model not only provides explicit size and geometry description of the protein molecule, but also offers a realistic means to characterise the adsorbed conformation in end-on (long axis perpendicular to collector surface) or side-on (short axis perpendicular to collector surface) (Yoon et al, 2003;Schrott et al, 2009).…”

Electrocatalytic oxidation of tyrosines shows signal enhancement in label-free protein biosensors

“…The catalyzed current of unfolded BSA was found to depend on the urea concentration, and the dependence was consistent with fluorescence measurement [105], which validated the EC method. Taking advantage of the unique properties of BDD electrodes, Einaga and co-workers monitored the conformation change of BSA by comparing its oxidation peak at a high potential of 1300 mV before and after addition of urea [67]. At least five redox-active species are involved in the oxidation reaction: Tyr, Tpr, Cys, Met, and disulfide bonds.…”

Investigation on the Conformation Change of Hemoglobin Immobilized on MPA-modified Electrode by Electrochemical Method

“…[8][9][10][11] Currently, electrochemical investigations on the folding/ unfolding of proteins mainly focus on metalloproteins, such as cytochrome c, myoglobin and hemoglobin (Hb). [12][13][14][15][16][17][18] The heme irons in these proteins are used as endogenous electroactive probes. 17,18 Proteins are commonly pre-unfolded by a certain denaturant of high concentration.…”